PROJECT SUMMARY The long-term objective of this proposal is to understand in quantitative detail the transduction mechanism in vertebrate olfactory receptor neurons (ORNs). During the current grant period, we have been able to resolve the response triggered by a single odorant-binding event on an ORN (the so- called unitary response). Surprisingly, we found that randomly encountered ORNs (and therefore randomly encountered odorant receptors, or ORs) all gave unitary responses of similar amplitudes, suggesting that an odorant-binding event has a very low probability of signaling downstream. In other words, the unitary response appears to reflect the effect of a single activated Golf/adenylyl cyclase complex. This phenomenon is apparently due to a very short odorant dwell-time on the OR molecule (i.e., rapid dissociation of the odorant from the OR). This short odorant dwell-time likewise dominates the termination of olfactory transduction. Hence, the traditionally believed determinants for terminating G-protein-coupled receptor (GPCR) signaling, namely, receptor phosphorylation and subsequent arrestin binding, are unimportant at least at the level of the unitary response, although they may still be important with intense and prolonged odorant stimulation. So far, the above results have been obtained from amphibian ORNs and in low-Ca2+ solution (in order to boost the unitary-response amplitude). In this application, we propose to continue experiments with amphibian ORNs but at the same time to use also mouse ORNs, which offer the distinct advantage of genetic manipulations. Aim 1 is to obtain the best estimate of the olfactory unitary-response amplitude in frog ORNs in physiological conditions (i.e., normal external Ca2+), to characterize its spatial spread along the olfactory cilium, to dissect its membrane-current components (cyclic-nucleotide-gated current versus Cl current), and to estimate the number of unitary responses required for bringing the ORN to firing threshold. Aim 2 is to launch a similar detailed study of the unitary response in mouse ORNs, which will serve as the groundwork for examining available genetic mouse lines for olfactory transduction. Aim 3 is to study/dissect Ca-dependent and Ca-independent adaptation by ORNs in more detail, largely by making use of genetic mouse lines. Aim 4 is to study constitutive OR activity in the absence of odorants, which so far has received little attention in vertebrate olfactory transduction. We have noticed this phenomenon in the course of previous experimentation. The experimental approach will involve predominantly suction-pipette recording from single ORNs of frog and WT or genetically engineered mice.